Friend or foe? Reciprocal regulation between E3 ubiquitin ligases and deubiquitinases

Protein ubiquitination is a post-translational modification that entails the covalent attachment of the small protein ubiquitin (Ub), which acts as a signal to direct protein stability, localization, or interactions. The Ub code is written by a family of enzymes called E3 Ub ligases (~600 members in humans), which can catalyze the transfer of either a single ubiquitin or the formation of a diverse array of polyubiquitin chains. This code can be edited or erased by a different set of enzymes termed deubiquitinases (DUBs; ~100 members in humans). While enzymes from these distinct families have seemingly opposing activities, certain E3-DUB pairings can also synergize to regulate vital cellular processes like gene expression, autophagy, innate immunity, and cell proliferation. In this review, we highlight recent studies describing Ub ligase-DUB interactions and focus on their relationships

Investigating the role of 53BP1 in regulating gene transcription.

53BP1 is a DNA damage responsive protein that plays a crucial role in checkpoint activation and DNA repair. In addition to its involvement in the cellular response to DNA damage, it has been suggested that 53BP1 can also function to regulate gene expression. 53BP1 was originally identified in a yeast two-hybrid screen for novel modulators of p53 transcriptional activity. Despite this, the role of 53BP1 in transcriptional regulation remains poorly understood. To investigate the effect of 53BP1 on cellular transcription, a microarray approach was utilised to study the gene expression patterns in cells treated with and without 53BP1 siRNA, before and after ionising radiation. Microarray analysis identified numerous genes whose expression was regulated by 53BP1 in the absence and presence of DNA damage. These data suggest that 53BP1 functions as a transcriptional regulator. In support of this, in vitro and in vivo studies have shown that 53BP1 binds to the transcriptional co-activators, CBP and p300. These findings indicate that the binding of 53BP1 to CBP and p300 may be facilitating its role as a regulator of transcription

Regulation of p53 by E3s

More than 40 years of research on p53 have given us tremendous knowledge about this protein. Today we know that p53 plays a role in different biological processes such as proliferation, invasion, pluripotency, metabolism, cell cycle control, ROS (reactive oxygen species) production, apoptosis, inflammation and autophagy. In the nucleus, p53 functions as a bona-fide transcription factor which activates and represses transcription of a number of target genes. In the cytoplasm, p53 can interact with proteins of the apoptotic machinery and by this also induces cell death. Despite being so important for the fate of the cell, expression levels of p53 are kept low in unstressed cells and the protein is largely inactive. The reason for the low expression level is that p53 is efficiently degraded by the ubiquitin-proteasome system and the vast inactivity of the tumor suppressor protein under normal growth conditions is due to the absence of activating and the presence of inactivating posttranslational modifications. E3s are important enzymes for these processes as they decorate p53 with ubiquitin and small ubiquitin-like proteins and by this control p53 degradation, stability and its subcellular localization. In this review, we provide an overview about E3s that target p53 and discuss the connection between p53, E3s and tumorigenesis

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.Eturauhassyöpä on miesten yleisimpiä syöpätyyppejä. Sen kehittyminen vaatii mieshormonialtistusta ja DNA-vaurioiden aiheuttamia muutoksia useissa solun kasvua säätelevien reittien toiminnassa. Viimeaikaisten tutkimusten mukaan virheet DNA:n kaksoissäiekatkosten korjauksessa voivat johtaa eturauhassyövässä tyypillisten geneettisten muutosten syntyyn. Ymmärrys näihin muutoksiin johtavista mekanismeista solussa auttaa eturauhassyövän hoidon kehittämisessä ja syövän ennaltaehkäisyssä. Eturauhanen ja seminaalivesikkelit kuuluvat miehen lisääntymiselimistöön, ja ne ovat rakenteellisesti ja toiminnallisesti samankaltaisia. Seminaalivesikkelikudoksen syöpä on kuitenkin äärimmäisen harvinainen, ja maailmanlaajuisesti on julkaistu vain noin 50 tapausta. Tässä työssä selvitimme, miten normaali eturauhanen ja seminaalivesikkeli tunnistavat DNA-vaurioita. Tätä tarkoitusta varten pystytimme uudenlaiset eturauhas- ja seminaalivesikkelikudosleikkeiden viljelymenetelmät, joihin aiheutimme DNA-vauriota säteilytyksellä tai kemikaaleilla. Näin pystyimme tutkimaan erilaistuneiden solutyyppien DNA-vauriovasteita. DNA:n kaksoissäiekatkokset käynnistivät tärkeän vauriontunnistusreitin molempien kudosten kaikissa pääsolutyypeissä. Yllättäen osa vauriosignaalia välittävistä proteiineista ei kuitenkaan toiminut samalla tavalla molempien kudosten kahdessa eri epiteelisolutyypissä. Lisäksi halusimme tutkia, miten eturauhasen ja seminaalivesikkelin epiteelisolut vastaavat DNA:n kaksoissäiekatkoksiin silloin kun ne jakautuvat aktiivisesti. Kudosleikemallissa jakautuvia soluja on vähän, joten selvitimme tätä kysymystä eturauhas- ja seminaalivesikkeliepiteelin primaarisolumalleissa. Primaarisolut eristetään potilasnäytteistä ja kasvatetaan solumaljoilla. Tietojemme mukaan tässä tutkimuksessa käytettiin ensimmäistä kertaa ihmisen seminaalivesikkeliepiteelin primaarisoluja. Aiheutimme sekä eturauhas- että seminaalivesikkelisoluihin kaksoissäiekatkoksia säteilytyksellä. Vastoin odotuksia havaitsimme, että eturauhasen epiteelisoluissa DNA-vaurio ei hidasta solunjakautumista, kun taas seminaalivesikkeliepiteelisoluissa solunjakautumisen hidastusvaste toimii. Tätä eroa voi selittää se, että seminaalivesikkelien epiteelisoluissa kaksi solunjakautumista DNA-vaurion jälkeen säätelevää proteiinia, p53 ja Wee1A, toimivat aktiivisemmin. Löydöksiemme perusteella on mahdollista, että eturauhasepiteeli on erityisen altis DNA-vaurioiden virheelliselle korjaukselle silloin, kun epiteelisolukko uudistuu, esimerkiksi tulehduksen aiheuttaman epiteelivaurion jälkeen

Ubiquitin E3 Ligases in Lung Disease

Diseases of the lung form the one of the largest causes of death globally. Inflammatory diseases such as Acute Respiratory Distress Syndrome, and fibrosis such as interstitial lung disease, in particular have high mortality rates with limited therapy. Thus, there is an unmet public health need for new avenues of intervention. Inflammatory, fibrotic, and nutrient lung diseases are driven by cellular signaling pathways, leading to pathological cell responses. Effector lung cells naturally dampen these deleterious signaling pathways; dysfunction of these dampening mechanisms play causal roles in lung disease, notably through excessive destruction of critical signal transduction proteins. Modulation of signal transduction protein degradation may have therapeutic effect by controlling deleterious signaling. The ubiquitin-proteasome system is the major cellular mechanism controlling protein degradation. Ubiquitin E3 ligase proteins are a critical part of ubiquitination, specifically targeting substrates for degradation. Research shows the importance of protein degradation in lung disease, however the potential to identify and inhibit specific E3-ligase-substrate interactions remains unexplored. Through both candidate-based and unbiased high-throughput screening techniques, we probed the importance of E3 ligases in lung disease through their targeted degradation of signal transduction proteins, and the therapeutic potential of E3 ligase inhibition. We investigated three aspects of lung disease – 1) inflammation and innate immunity, 2) fibrosis and interstitial lung disease, and 3) regulation of nutrient sensing mechanisms. Here we report multiple E3 ligase-substrate axes, including those associated with fibrosis: FIEL1-PIAS4; KLHL42-PPP2R5e, with innate immunity: PPP1R11-TLR2, RNF113A-CXCR4, KIAA0317-SOCS2, RNFT2-IL3Ra, and with nutrient sensing: RNF186-SESN2. We observed that E3 ligases potently control inflammatory signaling through control of cytokine receptors and signal modulators during acute inflammation and bacterial infection. We uncovered that E3 ligases are significantly associated with fibrotic signaling in interstitial lung fibrosis, and can be targeted by small molecules. Finally, we detected new mechanisms of nutrient sensor control leading to manipulation of anabolism. These results show the criticality of ubiquitin e3 ligases in the biology of lung inflammation, fibrosis, and nutrient sensing. Further, these studies validate ubiquitin E3 ligases as potential targets for therapeutic intervention to provide new tools to combat lung diseases

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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Molecular Targets of CNS Tumors

Molecular Targets of CNS Tumors is a selected review of Central Nervous System (CNS) tumors with particular emphasis on signaling pathway of the most common CNS tumor types. To develop drugs which specifically attack the cancer cells requires an understanding of the distinct characteristics of those cells. Additional detailed information is provided on selected signal pathways in CNS tumors